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Action of parathyroid extract on arterial blood pressure and on contraction and 45Ca exchange in isolated aorta of the rat
European Journal o f Pharmacology, 58 (1979) 163--167 G Elsevier/North-Holland Biomedical Press
163
A C T I O N O F P A R A T H Y R O I D E X T R A C T O N A R T E R I A L B L O O D P R E S S U R E A N D ON C O N T R A C T I O N A N D 4SCa E X C H A N G E IN I S O L A T E D A O R T A O F T H E R A T R. SCHLEIFFER, A. BERTHELOT and A. GAIRARD * Laboratoire de Physiologic, U.E.R. des Scierwes Pharmaceutiques, E.R.A. C.N.R.S. No. 787, 74, Route du Rhin, 67400 lllkirch Graffenstaden, France
Received 28 May 1979, accepted 7 June 1979
R. SCHLEIFFER, A. BERTHELOT and A. GAIRARD, Action o f parathyroid extract on arterial blood pressure and on contraction and 4sca exchange in isolated aorta o f the rat, European J. Pharmacol. 58 (1979) 163--167. The acute effects of parathyroid extract (PTE) on the cardiovascular system were the object of the study. PTE injected at concentrations of 10--100 U USP/kg into conscious rats induced a rapid, transitory fall in systolic blood pressure and a decrease in the total peripheral resistance. Since the cardiac output was unchanged, PTE must act on vascular smooth muscle. In vitro, PTE (2--16 U USP/ml) inhibited the tonic phase (which requires the presence of extracellular calcium) of the contraction of the thoracic aorta in response to phenylephrine or noradrenaline, asCa fluxes in the isolated aorta, measured by the lanthanum method, changed in the presence of a high concentration of PTE (1.0 U USP/ml). After 2 rain, the influx was lowered and the efflux was enhanced. These effects may have been specific, since they did not appear when PTE had been previously inactivated. Our results suggest that the vasodilator effect of PTE in the conscious rat is due to an action on Ca exchange in vascular smooth muscle. Parathyroid extract
4SCa exchange
Vascular system
1. I n t r o d u c t i o n * P a r a t h y r o i d e x t r a c t (PTE) s h o w e d a vasodilator e f f e c t in t h e a n e s t h e t i z e d d o g (Charb o n , 1 9 6 8 ) . A small d o s e (2 U USP/kg) increased t h e b l o o d flow t h r o u g h t h e coeliac a n d renal arteries. A similar e f f e c t was observed b y C h a r b o n a n d Hulstaert ( 1 9 7 4 ) , using a s y n t h e t i c p o l y p e p t i d e (1-34 p a r a t h y roid h o r m o n e (PTH)). More r e c e n t l y P T H was s h o w n t o have a positive c h r o n o t r o p i c a c t i o n on rat ventricle cells in c u l t u r e ( L a r n o et al., 1 9 7 8 ) . In t h e w o r k r e p o r t e d o n here, we have tried t o clarify t h e m e c h a n i s m o f t h e a c u t e e f f e c t o f PTE o n t h e cardiovascular s y s t e m , using e x p e r i m e n t s o n u n a n e s t h e t i z e d * Some results of this work were presented in a poster session of the IIIrd International Symposium on vascular neuroeffector mechanisms, July 24--26, 1978 (Louvain cn Woluv~-Wilrijk).
rats, isolated a o r t a e , and 4SCa exchanges in aortic tissue.
2. Materials a n d m e t h o d s T h e e x p e r i m e n t s were p e r f o r m e d o n male Sprague D a w l e y (SPF) rats weighing 2 0 0 - 3 0 0 g. T h e y were given f o o d a n d distilled w a t e r ad libitum. S u r g e r y was p e r f o r m e d u n d e r anesthesia with 30 m g / k g i n t r a p e r i t o n e a l s o d i u m p e n t o barbital. The b l o o d pressure ( B P ) w a s measured o n u n a n e s t h e t i z e d rats w i t h a S t a t h a m 23DPB p r o b e inserted via a p e r m a n e n t c a t h e t e r in t h e c a r o t i d artery. PTE or diluting fluid was a d m i n i s t e r e d via t h e jugular vein. The cardiac o u t p u t was d e t e r m i n e d b y therm o d i l u t i o n ( H o u d a s et al., 1 9 6 9 ) a n d t o t a l peripheral resistance was calculated ( A l e x a n d r e et al., 1 9 7 2 ) . F o r t h e studies o n a o r t a e , t h e
164 rats were decapitated under basal conditions and drained o f blood, and the aorta was quickly removed and placed in Krebs solution at 37°C. The vessel was then cut helically by the m e t h o d of Valette and Ba Muoi {1962} and incubated in Krebs solution at 37 ° C. PTE was added to the bath to a final concentration of 2--16 U USP/ml, 1 min before the addition of noradrenaline (NA) or phenylephrine (PE). 4SCa exchanges (influx and efflux} were evaluated in other experiments on isolated aorta by means of the " l a n t h a n u m m e t h o d " (Godfraind, 1976). The aortae were hemisected longitudinally, so that one part could be used as the control for the other part. In experiments on the influx of 4SCa into the Laresistant Ca fraction of aorta, the aortic strips were preincubated for 2 h in Krebs solution. After 5 min exposure to Krebs solution containing 4SCa (0.4 pCi/ml), they were placed for 2 or 10 min in tile same 4SCa medium also containing 0.1 or 1.0 U USP PTE/ml or none, then transferred to a 50 mM La solution for 5 min. For measurement of the 4SCa efflux the strips were preincubated for 2 h in 4~Ca Krebs solution. They were placed for 5 min in nonradioactive Krebs solution then transferred to the same medium w i t h o u t or with 0.1 or 1.0 U USP PTE/ml. They were then washed in the 50 mM La solution. Each strip was weighed and digested by the addition of 50 pl of perchloric acid (37% w/v) and 50 ttl of H202 (30 vol) at 75°C for 15 min. Aortic 4SCa was expressed as mmol/kg wet weight tissue. The specificity of the effect of PTE disappeared when the h o r m o n e had been inactivated by trypsin (2% trypsin at pH 8) at 37°C for 8 h. The following reagents were used: lyophilized bovine PTE, Eli-Lilly (17 U USP/mg, lot No. RG 76); trypsin, Choay; L-phenylephrine (PE) Neosynephrine ® from Badrial; L-noradrenaline (NA} from Flucka AG; 4SCaCl: from the Radiochemical Centre, Amersham; scintillation solution, Aqualuma® from Lumac, for estimation of the 4SCa radioactivity; two different Krebs solutions, con-
R. SCHLEIFFER ET AL. taining (mM) (1) 115NaCl, 4.6KC1, 25 NaHCO3, 1 KH2PO~, 1.4 MgSO4 • 7 H20, 2.5 CaC12 and 10 glucose (pH 7.4) for the measurement of the contraction of the isolated aorta, and (2) 122 NaC1, 5.9 KC1, 15 NaHCO~, 1.25 MgC12, 1.25 CaCl: and 11 glucose (pH 7.2), for the measurement of the ~SCa fluxes (both media were continuously bubbled with 95% 02 + 5% CO2 at 37°C; and a lanthanum solution containing (raM) 122 NaCl, 5.9 KC1, 1.25 MgCl2, 11 glucose, and 50 LaCl.~ (pH 6.8). The results are expressed as means + S.E.M., and statistical comparisons were made using Student's t-test for paired values; regression curves were calculated for the experiment with graded doses of PTE.
3. Results 3.1. A c t i o n o f PTE on B P
A single intravenous injection of PTE (100 U USP/kg) in the unanesthetized rat induced a rapid but transitory fall in BP (--23 mm Hg after 3 min, the initial BP was not restored after 9 min} (fig. 1). At the third min after PTE injection, when the hypotension was maximal, the cardiac o u t p u t remained unchanged (before PTE, 197 + 6; after PTE, 197 -+ 5 ml × min -~ × kg-~), and the total peripheral resistance was significantly decreased (before PTE, 588 + 28; after PTE, 471 + 28 dynes × sec -~ X cm -s × kg -2 X 102; P < 0.01). The fall in BP became more pronounced when increasingly large doses of PTE were administered every 2 min (fig. 2). A clear-cut dose-response curve was obtained between 10 and 160 U USP/kg (y = --21.7 log x + 147.4}. 3.2. A c t i o n o f P T E on isolated aorta
the contraction
of
The analysis of the contraction induced under isometric conditions by NA or PE permitted two components to be dif-
165
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Fig. 1. Effect of a single intravenous injection of PTE (100 U USP/kg) on the systolic blood pressure (BP), cardiac o u t p u t , and total peripheral resistance in the conscious rat. The values are the means-+ S.E.M. S t u d e n t ' s t-test for paired observations was used for statistical analyses (* P < 0.05, ** P < 0.01 vs. the value from just before the injection of PTE; n = 6). Plain blocks a, cardiac o u t p u t (ml × rain-! × kg -1); dashed blocks ~, total peripheral resistance (dynes × see -1 X cm -$ X kg -2 x 100).
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ferentiated (fig. 3A). The first c o m p o n e n t (phasic phase (P)) was very fast (about 5 sec) and was followed by a slow c o m p o n e n t (tonic phase (T)) which lasted 5 min. (These two phases were estimated by graphic resolution). PTE decreased the m a x i m u m tension only in the tonic phase (which is dependent on extracellular Ca) of the contraction of isolated aorta in response to an a-stimulant (fig. 3). It did not change the fast phase. This effect was dose
Fig. 6 shows the effect of PTE on 4SCa exchanges in isolated aorta. PTE changed the influx and the efflux of 4SCa in the lanthanum-resistant Ca fraction. In aortas which had been incubated for 2 min in a solution containing PTE (0.1 U USP/ml), the influx of 4SCa was significantly enhanced (+0.025 mmol/kg wet wt; P < 0.05); this result was reversed with the higher concentration, 1.0 U USP/ml (--0.025 mmol/kg wet wt; P < 0.05). At the same time {2 rain) the efflux was unchanged with the low dose (0.1 U USP/ ml) and increased with 1.0 U USP/ml (+0.036
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Fig. 2. Cumulative dose-response curve of the decrease in systolic b l o o d pressure (BP) with injections of graded doses of PTE in the conscious rat. BP was measured just b e f o r e each injection of PTE, which was given at 2 rain intervals. The values are the means ± S.E.M. S t u d e n t ' s t-test for paired observations was used for statistical analyses ( * P < 0.05, ** P < 0.001 vs. the value f r o m just before the first injection of P T E ; n = 6) (y = - - 2 1 . 7 log x + 147.4, r = 0.98).
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Fig. 3. E x a m p l e o f the effect of P T E on the contract i o n o f an isolated aorta in response to phenylephrine (PE) w i t h o u t PTE ( A ) a n d with PTE ( B ; 4 U USP/ml). Phasic phase (P) and tonic phase (T). Tension is expressed in terms of the a m o u n t of weight lifted by the force o f the contraction.
166
R. S C H L E I F F E R ET AL.
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Fig. 4. C u m u l a t i v e log d o s e - r e s p o n s e curves o f t h e increase in t h e m a x i m a l c o n t r a c t i o n o f t h e isolated a o r t a w i t h graded doses o f p h e n y l e p h r i n e (PE) witho u t ( c o n t r o l s ) a n d w i t h P T E (2 to 16 U U S P / m l ) . T h e m a x i m u m t e n s i o n in c o n t r o l s ( t a k e n as 1 0 0 % ) was o b t a i n e d w i t h PE a l o n e , at a c o n c e n t r a t i o n o f 100 × 10 -7 m m o l / m l (n = 6). T h e small g r a p h at the b o t t o m s h o w s a n e x a m p l e o f a c u m u l a t i v e doseresponse curve with phenylephrine. Analysis of v a r i a n c e s h o w e d t h e significance o f t h e d i f f e r e n c e s between control and PTE-treated groups to be P < 0.01 for 2, a n d P < 0.001 for 4, 8, 1 6 U U S P PTE/ml.
Fig. 6. 4SCa c o n t e n t o f t h e l a n t h a n u m - r e s i s t a n t Ca f r a c t i o n o f isolated a o r t a ( m m o l / k g wet weight) 2 a n d ] 0 m i n a f t e r i n c u b a t i o n w i t h 0.1 ( A ) or 1.0 (B) U USP P T E / m l . T h e n u m b e r o f a o r t a s is i n d i c a t e d w i t h i n each c o l u m n (0 = t h e a m o u n t o f 4SCa in cont r o l s i n c u b a t e d w i t h o u t PTE). ~ Dashed b l o c k s 2 rain, s s t i p p l e d b l o c k s 10 rain. * P < 0 . 0 5 , ** P < 0.01.
mmol/ml). content of aorta (fig. incubation,
Discussion
This effect decreased the 45Ca the La-resistant fraction in the 6, efflux, B). After 10 min of there was no significant change in
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NA | m mol/ml) Fig. 5. C u m u l a t i v e log d o s e - r e s p o n s e curves o f t h e increase in t h e m a x i m a l c o n t r a c t i o n o f t h e isolated a o r t a with graded doses o f n o r a d r e n a l i n e ( N A ) witho u t ( c o n t r o l s ) a n d w i t h P T E (9 U U S P / m l ) . T h e m a x i m a l t e n s i o n in c o n t r o l s ( t a k e n as 100%) was o b t a i n e d w i t h N A a l o n e , at a c o n c e n t r a t i o n o f 100 x 10 -7 m m o l / m l (n = 6). Significance o f t h e d i f f e r e n c e s b e t w e e n c o n t r o l a n d P T E - t r e a t e d groups, P < 0.001 (analysis of variance).
4s Ca flux. Again, these effects must have been specific, because inactivation of the PTE abolished them.
The present study confirms for the conscious rat Charbon's observation (1968) of the vasodilator effect of PTE in the anesthetized dog. A hypotensive action similar to that o f PTE was also observed by Charbon and Hulstaert (1974) after administration of the synthetic peptide 1-34 PTH. Since cardiac o u t p u t was unchanged and total peripheral resistance decreased in our experiments, PTE must act on vascular resistance. The study of 4SCa exchanges in the isolated aorta by the lanthanum m e t h o d may explain the vasodilator effect of PTE in vivo. According to Freeman and Daniel (1973), and Burton and Godfraind {1974), the lanthanum-resistant Ca fraction approximates the intracellular Ca. After administration of a high dose of PTE, we found a decrease in the influx of Ca into the lanthanum-resistant Ca fraction and an increase in the efflux of this fraction which probably decreased the total pool of vascular Ca.
PARATHYROID EXTRACT AND THE VASCULAR SYSTEM
These observations are in agreement with those of Parsons and Robinson (1971), who showed a decrease in the 47Ca content of noncalcified tissues (muscle, skin, liver, gut, kidney) of dogs 10 min after simultaneous i.v. injection of radiocalcium and PTH (30 U USP/dog). On the other hand, in contrast to previous reports, lower concentrations of PTE (0.1 U USP/ml) increased the influx of 4SCa into the lanthanum-resistant Ca fraction of aorta. Borle and Uchikawa (1978) also found that highly purified PTH (15 mU/ml) augmented the pool of exchangeable Ca and stimulated the flow of Ca into cultured renal cells. Similarly, in non~alcified tissues PTE has been shown to rapidly increase the amount of Ca in HeLa cells (Borle, 1968), and renal cells (Borle, 1970). Our data on aortic contraction due to one of the a-stimulants, PE (which acts selectively on the a-receptors), or NA (which is the physiological neurotransmitter), served to show that PTE inhibited only the tonic phase. This part of the contraction requires the presence of extracellar Ca (Godfraind and Kaba, 1972; Van Breemen et al. 1972). It probably means that PTE inhibits the influx of Ca during the tonic phase of the aortic contraction induced b y an a-stimulant and additional data are necessary before a definite conclusion can be reached. Fluxes of 4SCa in aortic vascular smooth muscle of the rat may nevertheless explain the vasodilator effect of PTE observed in our experiments and those described by Charbon (1968) for the dog.
Acknowledgements We are grateful to Prof. T. Godfraind, Universit4 Catholique de Louvain, Brussels, Belgium, who kindly taught us to use the lathanum technique. We also thank Messrs. J. Ketz and P. Widemann for skilful technical assistance. Bovine parathyroid extract specially lyophilized for us was a generous gift of Eli-Lilly. This work was supported b y INSERM (No. 3276 64002) and DGRST (A.T. No. 77.7 1391).
167
References Alexandre, J.M., J. Menard, C. Chevillard and H. Schmitt, 1972, Etude h4modynamique de l'hypertension 4s~rinique chez le rat, J. Pharmacol. 2, 143. Borle, A.B., 1968, Calcium metabolism in HeLa cells and the effects of parathyroid hormone, J. Cell Biol. 3 6 , 5 6 7 . Borle, A.B., 1970, Kenetic analyses of calcium movements in cell cultures. III. Effects of calcium and parathyroid hormone in kidney cells, J. Gen. Physiol. 5 5 , 1 6 3 . Borle, A.B. and T. Uchikawa, 1978, Effects of parathyroid hormone on the distribution and transport of calcium in cultured kidney cells, Endocrinology 102, 1725. Burton, J. and T. Godfraind, 1974, Sodium--calcium sites in smooth muscle and their accessibility to lanthanum, J. Physiol. 2 4 1 , 2 8 7 . Ch.arbon, G.A., 1968, A rapid and selective vasodilatator effect of parathyroid hormone, European J. Pharmacol. 3 , 2 7 5 . Charbon, G.A. and P.F. Hulstaert, 1974, Augmentation of arterial hepatic and renal flow b y extracted and synthetic parathyroid hormone, Endocrinology 9 5 , 6 2 1 . Freeman, D.J. and E.E. Daniel, 1973, Calcium movement in vascular smooth muscle and its detection using lanthanum as a tool, Can. J. Physiol. Pharmacol. 5 1 , 9 0 0 . Godfraind, T., 1976, Calcium exchange in vascular smooth muscle, action of noradrenaline and lanthanum, J. Physiol. 260, 21. Godfraind, T. and A. Kaba, 1972, The role of calcium in the action of drugs on vascular smooth muscle, Arch. Intern. Pharmacodyn. Therap. 196, 35. Houdas, Y., J.Y. Ketelers, C. Houdas-Heyraud and E. Lerche, 1969, La thermodilution appliqu~e ~ la mesure de d~bit cardiaque des petits animaux de laboratoire, Pathol. Biol. 17, 1069. Larno, S., F. Lhoste, M.C. Auclair and P. Lechat, 1978, Interaction between parathyroid hormone and the ~-adrenoreceptor system in cultured rat myocardial cells, Abstracts 7th International Congress of Pharmacology, Paris, p. 148. Parsons, J.A. and C.J. Robinson, 1971, Calcium shift into bone causing transient hypocalcaemia after injection o f parathyroid hormone, Nature 230, 581. Valette, G. and N. Ba Muoi, 1962, De l'emploi de segments d'aorte de rats pour le titrage de solutions d'adr4naline, Ann. Pharmacol. Fran. 20, 577. Van Breemen, C., B. Farinas, P. Gerba and E. McNaughton, 1972, Excitation--contraction coupling in arterial smooth muscle b y the " L a - m e t h o d " for measuring cellular calcium influx, Circulation Res. 30, 44.
163
A C T I O N O F P A R A T H Y R O I D E X T R A C T O N A R T E R I A L B L O O D P R E S S U R E A N D ON C O N T R A C T I O N A N D 4SCa E X C H A N G E IN I S O L A T E D A O R T A O F T H E R A T R. SCHLEIFFER, A. BERTHELOT and A. GAIRARD * Laboratoire de Physiologic, U.E.R. des Scierwes Pharmaceutiques, E.R.A. C.N.R.S. No. 787, 74, Route du Rhin, 67400 lllkirch Graffenstaden, France
Received 28 May 1979, accepted 7 June 1979
R. SCHLEIFFER, A. BERTHELOT and A. GAIRARD, Action o f parathyroid extract on arterial blood pressure and on contraction and 4sca exchange in isolated aorta o f the rat, European J. Pharmacol. 58 (1979) 163--167. The acute effects of parathyroid extract (PTE) on the cardiovascular system were the object of the study. PTE injected at concentrations of 10--100 U USP/kg into conscious rats induced a rapid, transitory fall in systolic blood pressure and a decrease in the total peripheral resistance. Since the cardiac output was unchanged, PTE must act on vascular smooth muscle. In vitro, PTE (2--16 U USP/ml) inhibited the tonic phase (which requires the presence of extracellular calcium) of the contraction of the thoracic aorta in response to phenylephrine or noradrenaline, asCa fluxes in the isolated aorta, measured by the lanthanum method, changed in the presence of a high concentration of PTE (1.0 U USP/ml). After 2 rain, the influx was lowered and the efflux was enhanced. These effects may have been specific, since they did not appear when PTE had been previously inactivated. Our results suggest that the vasodilator effect of PTE in the conscious rat is due to an action on Ca exchange in vascular smooth muscle. Parathyroid extract
4SCa exchange
Vascular system
1. I n t r o d u c t i o n * P a r a t h y r o i d e x t r a c t (PTE) s h o w e d a vasodilator e f f e c t in t h e a n e s t h e t i z e d d o g (Charb o n , 1 9 6 8 ) . A small d o s e (2 U USP/kg) increased t h e b l o o d flow t h r o u g h t h e coeliac a n d renal arteries. A similar e f f e c t was observed b y C h a r b o n a n d Hulstaert ( 1 9 7 4 ) , using a s y n t h e t i c p o l y p e p t i d e (1-34 p a r a t h y roid h o r m o n e (PTH)). More r e c e n t l y P T H was s h o w n t o have a positive c h r o n o t r o p i c a c t i o n on rat ventricle cells in c u l t u r e ( L a r n o et al., 1 9 7 8 ) . In t h e w o r k r e p o r t e d o n here, we have tried t o clarify t h e m e c h a n i s m o f t h e a c u t e e f f e c t o f PTE o n t h e cardiovascular s y s t e m , using e x p e r i m e n t s o n u n a n e s t h e t i z e d * Some results of this work were presented in a poster session of the IIIrd International Symposium on vascular neuroeffector mechanisms, July 24--26, 1978 (Louvain cn Woluv~-Wilrijk).
rats, isolated a o r t a e , and 4SCa exchanges in aortic tissue.
2. Materials a n d m e t h o d s T h e e x p e r i m e n t s were p e r f o r m e d o n male Sprague D a w l e y (SPF) rats weighing 2 0 0 - 3 0 0 g. T h e y were given f o o d a n d distilled w a t e r ad libitum. S u r g e r y was p e r f o r m e d u n d e r anesthesia with 30 m g / k g i n t r a p e r i t o n e a l s o d i u m p e n t o barbital. The b l o o d pressure ( B P ) w a s measured o n u n a n e s t h e t i z e d rats w i t h a S t a t h a m 23DPB p r o b e inserted via a p e r m a n e n t c a t h e t e r in t h e c a r o t i d artery. PTE or diluting fluid was a d m i n i s t e r e d via t h e jugular vein. The cardiac o u t p u t was d e t e r m i n e d b y therm o d i l u t i o n ( H o u d a s et al., 1 9 6 9 ) a n d t o t a l peripheral resistance was calculated ( A l e x a n d r e et al., 1 9 7 2 ) . F o r t h e studies o n a o r t a e , t h e
164 rats were decapitated under basal conditions and drained o f blood, and the aorta was quickly removed and placed in Krebs solution at 37°C. The vessel was then cut helically by the m e t h o d of Valette and Ba Muoi {1962} and incubated in Krebs solution at 37 ° C. PTE was added to the bath to a final concentration of 2--16 U USP/ml, 1 min before the addition of noradrenaline (NA) or phenylephrine (PE). 4SCa exchanges (influx and efflux} were evaluated in other experiments on isolated aorta by means of the " l a n t h a n u m m e t h o d " (Godfraind, 1976). The aortae were hemisected longitudinally, so that one part could be used as the control for the other part. In experiments on the influx of 4SCa into the Laresistant Ca fraction of aorta, the aortic strips were preincubated for 2 h in Krebs solution. After 5 min exposure to Krebs solution containing 4SCa (0.4 pCi/ml), they were placed for 2 or 10 min in tile same 4SCa medium also containing 0.1 or 1.0 U USP PTE/ml or none, then transferred to a 50 mM La solution for 5 min. For measurement of the 4SCa efflux the strips were preincubated for 2 h in 4~Ca Krebs solution. They were placed for 5 min in nonradioactive Krebs solution then transferred to the same medium w i t h o u t or with 0.1 or 1.0 U USP PTE/ml. They were then washed in the 50 mM La solution. Each strip was weighed and digested by the addition of 50 pl of perchloric acid (37% w/v) and 50 ttl of H202 (30 vol) at 75°C for 15 min. Aortic 4SCa was expressed as mmol/kg wet weight tissue. The specificity of the effect of PTE disappeared when the h o r m o n e had been inactivated by trypsin (2% trypsin at pH 8) at 37°C for 8 h. The following reagents were used: lyophilized bovine PTE, Eli-Lilly (17 U USP/mg, lot No. RG 76); trypsin, Choay; L-phenylephrine (PE) Neosynephrine ® from Badrial; L-noradrenaline (NA} from Flucka AG; 4SCaCl: from the Radiochemical Centre, Amersham; scintillation solution, Aqualuma® from Lumac, for estimation of the 4SCa radioactivity; two different Krebs solutions, con-
R. SCHLEIFFER ET AL. taining (mM) (1) 115NaCl, 4.6KC1, 25 NaHCO3, 1 KH2PO~, 1.4 MgSO4 • 7 H20, 2.5 CaC12 and 10 glucose (pH 7.4) for the measurement of the contraction of the isolated aorta, and (2) 122 NaC1, 5.9 KC1, 15 NaHCO~, 1.25 MgC12, 1.25 CaCl: and 11 glucose (pH 7.2), for the measurement of the ~SCa fluxes (both media were continuously bubbled with 95% 02 + 5% CO2 at 37°C; and a lanthanum solution containing (raM) 122 NaCl, 5.9 KC1, 1.25 MgCl2, 11 glucose, and 50 LaCl.~ (pH 6.8). The results are expressed as means + S.E.M., and statistical comparisons were made using Student's t-test for paired values; regression curves were calculated for the experiment with graded doses of PTE.
3. Results 3.1. A c t i o n o f PTE on B P
A single intravenous injection of PTE (100 U USP/kg) in the unanesthetized rat induced a rapid but transitory fall in BP (--23 mm Hg after 3 min, the initial BP was not restored after 9 min} (fig. 1). At the third min after PTE injection, when the hypotension was maximal, the cardiac o u t p u t remained unchanged (before PTE, 197 + 6; after PTE, 197 -+ 5 ml × min -~ × kg-~), and the total peripheral resistance was significantly decreased (before PTE, 588 + 28; after PTE, 471 + 28 dynes × sec -~ X cm -s × kg -2 X 102; P < 0.01). The fall in BP became more pronounced when increasingly large doses of PTE were administered every 2 min (fig. 2). A clear-cut dose-response curve was obtained between 10 and 160 U USP/kg (y = --21.7 log x + 147.4}. 3.2. A c t i o n o f P T E on isolated aorta
the contraction
of
The analysis of the contraction induced under isometric conditions by NA or PE permitted two components to be dif-
165
P A R A T H Y R O I D E X T R A C T A N D T H E V A S C U L A R SYSTEM 140
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Fig. 1. Effect of a single intravenous injection of PTE (100 U USP/kg) on the systolic blood pressure (BP), cardiac o u t p u t , and total peripheral resistance in the conscious rat. The values are the means-+ S.E.M. S t u d e n t ' s t-test for paired observations was used for statistical analyses (* P < 0.05, ** P < 0.01 vs. the value from just before the injection of PTE; n = 6). Plain blocks a, cardiac o u t p u t (ml × rain-! × kg -1); dashed blocks ~, total peripheral resistance (dynes × see -1 X cm -$ X kg -2 x 100).
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ferentiated (fig. 3A). The first c o m p o n e n t (phasic phase (P)) was very fast (about 5 sec) and was followed by a slow c o m p o n e n t (tonic phase (T)) which lasted 5 min. (These two phases were estimated by graphic resolution). PTE decreased the m a x i m u m tension only in the tonic phase (which is dependent on extracellular Ca) of the contraction of isolated aorta in response to an a-stimulant (fig. 3). It did not change the fast phase. This effect was dose
Fig. 6 shows the effect of PTE on 4SCa exchanges in isolated aorta. PTE changed the influx and the efflux of 4SCa in the lanthanum-resistant Ca fraction. In aortas which had been incubated for 2 min in a solution containing PTE (0.1 U USP/ml), the influx of 4SCa was significantly enhanced (+0.025 mmol/kg wet wt; P < 0.05); this result was reversed with the higher concentration, 1.0 U USP/ml (--0.025 mmol/kg wet wt; P < 0.05). At the same time {2 rain) the efflux was unchanged with the low dose (0.1 U USP/ ml) and increased with 1.0 U USP/ml (+0.036
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Fig. 2. Cumulative dose-response curve of the decrease in systolic b l o o d pressure (BP) with injections of graded doses of PTE in the conscious rat. BP was measured just b e f o r e each injection of PTE, which was given at 2 rain intervals. The values are the means ± S.E.M. S t u d e n t ' s t-test for paired observations was used for statistical analyses ( * P < 0.05, ** P < 0.001 vs. the value f r o m just before the first injection of P T E ; n = 6) (y = - - 2 1 . 7 log x + 147.4, r = 0.98).
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166
R. S C H L E I F F E R ET AL.
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Fig. 4. C u m u l a t i v e log d o s e - r e s p o n s e curves o f t h e increase in t h e m a x i m a l c o n t r a c t i o n o f t h e isolated a o r t a w i t h graded doses o f p h e n y l e p h r i n e (PE) witho u t ( c o n t r o l s ) a n d w i t h P T E (2 to 16 U U S P / m l ) . T h e m a x i m u m t e n s i o n in c o n t r o l s ( t a k e n as 1 0 0 % ) was o b t a i n e d w i t h PE a l o n e , at a c o n c e n t r a t i o n o f 100 × 10 -7 m m o l / m l (n = 6). T h e small g r a p h at the b o t t o m s h o w s a n e x a m p l e o f a c u m u l a t i v e doseresponse curve with phenylephrine. Analysis of v a r i a n c e s h o w e d t h e significance o f t h e d i f f e r e n c e s between control and PTE-treated groups to be P < 0.01 for 2, a n d P < 0.001 for 4, 8, 1 6 U U S P PTE/ml.
Fig. 6. 4SCa c o n t e n t o f t h e l a n t h a n u m - r e s i s t a n t Ca f r a c t i o n o f isolated a o r t a ( m m o l / k g wet weight) 2 a n d ] 0 m i n a f t e r i n c u b a t i o n w i t h 0.1 ( A ) or 1.0 (B) U USP P T E / m l . T h e n u m b e r o f a o r t a s is i n d i c a t e d w i t h i n each c o l u m n (0 = t h e a m o u n t o f 4SCa in cont r o l s i n c u b a t e d w i t h o u t PTE). ~ Dashed b l o c k s 2 rain, s s t i p p l e d b l o c k s 10 rain. * P < 0 . 0 5 , ** P < 0.01.
mmol/ml). content of aorta (fig. incubation,
Discussion
This effect decreased the 45Ca the La-resistant fraction in the 6, efflux, B). After 10 min of there was no significant change in
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NA | m mol/ml) Fig. 5. C u m u l a t i v e log d o s e - r e s p o n s e curves o f t h e increase in t h e m a x i m a l c o n t r a c t i o n o f t h e isolated a o r t a with graded doses o f n o r a d r e n a l i n e ( N A ) witho u t ( c o n t r o l s ) a n d w i t h P T E (9 U U S P / m l ) . T h e m a x i m a l t e n s i o n in c o n t r o l s ( t a k e n as 100%) was o b t a i n e d w i t h N A a l o n e , at a c o n c e n t r a t i o n o f 100 x 10 -7 m m o l / m l (n = 6). Significance o f t h e d i f f e r e n c e s b e t w e e n c o n t r o l a n d P T E - t r e a t e d groups, P < 0.001 (analysis of variance).
4s Ca flux. Again, these effects must have been specific, because inactivation of the PTE abolished them.
The present study confirms for the conscious rat Charbon's observation (1968) of the vasodilator effect of PTE in the anesthetized dog. A hypotensive action similar to that o f PTE was also observed by Charbon and Hulstaert (1974) after administration of the synthetic peptide 1-34 PTH. Since cardiac o u t p u t was unchanged and total peripheral resistance decreased in our experiments, PTE must act on vascular resistance. The study of 4SCa exchanges in the isolated aorta by the lanthanum m e t h o d may explain the vasodilator effect of PTE in vivo. According to Freeman and Daniel (1973), and Burton and Godfraind {1974), the lanthanum-resistant Ca fraction approximates the intracellular Ca. After administration of a high dose of PTE, we found a decrease in the influx of Ca into the lanthanum-resistant Ca fraction and an increase in the efflux of this fraction which probably decreased the total pool of vascular Ca.
PARATHYROID EXTRACT AND THE VASCULAR SYSTEM
These observations are in agreement with those of Parsons and Robinson (1971), who showed a decrease in the 47Ca content of noncalcified tissues (muscle, skin, liver, gut, kidney) of dogs 10 min after simultaneous i.v. injection of radiocalcium and PTH (30 U USP/dog). On the other hand, in contrast to previous reports, lower concentrations of PTE (0.1 U USP/ml) increased the influx of 4SCa into the lanthanum-resistant Ca fraction of aorta. Borle and Uchikawa (1978) also found that highly purified PTH (15 mU/ml) augmented the pool of exchangeable Ca and stimulated the flow of Ca into cultured renal cells. Similarly, in non~alcified tissues PTE has been shown to rapidly increase the amount of Ca in HeLa cells (Borle, 1968), and renal cells (Borle, 1970). Our data on aortic contraction due to one of the a-stimulants, PE (which acts selectively on the a-receptors), or NA (which is the physiological neurotransmitter), served to show that PTE inhibited only the tonic phase. This part of the contraction requires the presence of extracellar Ca (Godfraind and Kaba, 1972; Van Breemen et al. 1972). It probably means that PTE inhibits the influx of Ca during the tonic phase of the aortic contraction induced b y an a-stimulant and additional data are necessary before a definite conclusion can be reached. Fluxes of 4SCa in aortic vascular smooth muscle of the rat may nevertheless explain the vasodilator effect of PTE observed in our experiments and those described by Charbon (1968) for the dog.
Acknowledgements We are grateful to Prof. T. Godfraind, Universit4 Catholique de Louvain, Brussels, Belgium, who kindly taught us to use the lathanum technique. We also thank Messrs. J. Ketz and P. Widemann for skilful technical assistance. Bovine parathyroid extract specially lyophilized for us was a generous gift of Eli-Lilly. This work was supported b y INSERM (No. 3276 64002) and DGRST (A.T. No. 77.7 1391).
167
References Alexandre, J.M., J. Menard, C. Chevillard and H. Schmitt, 1972, Etude h4modynamique de l'hypertension 4s~rinique chez le rat, J. Pharmacol. 2, 143. Borle, A.B., 1968, Calcium metabolism in HeLa cells and the effects of parathyroid hormone, J. Cell Biol. 3 6 , 5 6 7 . Borle, A.B., 1970, Kenetic analyses of calcium movements in cell cultures. III. Effects of calcium and parathyroid hormone in kidney cells, J. Gen. Physiol. 5 5 , 1 6 3 . Borle, A.B. and T. Uchikawa, 1978, Effects of parathyroid hormone on the distribution and transport of calcium in cultured kidney cells, Endocrinology 102, 1725. Burton, J. and T. Godfraind, 1974, Sodium--calcium sites in smooth muscle and their accessibility to lanthanum, J. Physiol. 2 4 1 , 2 8 7 . Ch.arbon, G.A., 1968, A rapid and selective vasodilatator effect of parathyroid hormone, European J. Pharmacol. 3 , 2 7 5 . Charbon, G.A. and P.F. Hulstaert, 1974, Augmentation of arterial hepatic and renal flow b y extracted and synthetic parathyroid hormone, Endocrinology 9 5 , 6 2 1 . Freeman, D.J. and E.E. Daniel, 1973, Calcium movement in vascular smooth muscle and its detection using lanthanum as a tool, Can. J. Physiol. Pharmacol. 5 1 , 9 0 0 . Godfraind, T., 1976, Calcium exchange in vascular smooth muscle, action of noradrenaline and lanthanum, J. Physiol. 260, 21. Godfraind, T. and A. Kaba, 1972, The role of calcium in the action of drugs on vascular smooth muscle, Arch. Intern. Pharmacodyn. Therap. 196, 35. Houdas, Y., J.Y. Ketelers, C. Houdas-Heyraud and E. Lerche, 1969, La thermodilution appliqu~e ~ la mesure de d~bit cardiaque des petits animaux de laboratoire, Pathol. Biol. 17, 1069. Larno, S., F. Lhoste, M.C. Auclair and P. Lechat, 1978, Interaction between parathyroid hormone and the ~-adrenoreceptor system in cultured rat myocardial cells, Abstracts 7th International Congress of Pharmacology, Paris, p. 148. Parsons, J.A. and C.J. Robinson, 1971, Calcium shift into bone causing transient hypocalcaemia after injection o f parathyroid hormone, Nature 230, 581. Valette, G. and N. Ba Muoi, 1962, De l'emploi de segments d'aorte de rats pour le titrage de solutions d'adr4naline, Ann. Pharmacol. Fran. 20, 577. Van Breemen, C., B. Farinas, P. Gerba and E. McNaughton, 1972, Excitation--contraction coupling in arterial smooth muscle b y the " L a - m e t h o d " for measuring cellular calcium influx, Circulation Res. 30, 44.